1. Field of the Invention
This invention relates to methods of tracking cells in vivo and methods for determining in vivo cell lifetime.
2. Background Information
In vivo cellular tracking and lifetime determination require that cells be labelled with a marker that is stable in the cell, i.e., is not lost from the cell, and that does not significantly affect cell function. Presently available markers fail to provide the necessary characteristics. Fluorescent antibodies, for example, are not suitable because they readily dissociate from the cells. Other potential markers cannot be used because they interfere with cell function.
Cyanine dyes have been used in various biological applications. Dioxacarbocyanine dyes have been used in performing white blood cell differential counts. Gunter Valet, Max planck Ges Wissensch; Patent Accession Number 84-102307/17, Simultaneous Quantitative Determination of Blood Cells by Selective Staining and Measuring Volume and Fluorescence. The dyes utilized in these studies, however, are short chain carbocyanine dyes (less than ten carbons) and respond to changes in membrane potentials. Furthermore, the short chain carbocyanine dyes enter the cells mitochondria, are cytotoxic, and when the cells are washed these dyes easily leak out of the cell whether or not the membrane potential of the cell is changed. Other short aliphatic chain cyanine dyes are used in many other biological assays. The short chain molecules, however, respond to membrane potentials and cross the cell membrane, penetrating into the mitochondria. H. M. Shapiro, U.S. Pat. No. 4,343,782, Aug. 10, 1982. The short chain dyes also are toxic to cells and cannot be used to track cells in vivo.
Tricarbocyanine dYes (Fox, I. J., et al., Proc. Mayo Clinic, 32:478-484, 1957 ) and Evans-Blue dye (Schad, H., et al., Pfluegers Arch. Eur. J. Physiol., 370(2):139-144, 1977) have been used in vivo to estimate cardiac output by a dilution method. Dow (Dow, P., Physiol. Rev., 36:77-102, 1956) describes the method as injection of a known amount of some intravascular indicator on the venus side of the lungs, and measurement of the time course of arterial concentration of the indicator to determine the volume between the points of injection and sampling. These dyes are not used to stain cells.